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Biomedical subjects

A T Look

Publications and source records attributed to A T Look.

At least 91 records · Page 5Linked to original sources
PubMed1995

E2A-HLF-mediated cell transformation requires both the trans-activation domains of E2A and the leucine zipper dimerization domain of HLF.

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) translocation in childhood acute pro-B-cell leukemia, encodes a hybrid protein that contains the paired trans-activation domains of E2A (E12/E47) linked to the basic region/leucine zipper DNA-binding and dimerization domain of hepatic leukemia factor (HLF). To assess the transforming potential of this novel gene, we introduced it into NIH 3T3 murine fibroblasts by using an expression vector that also contained the neomycin resistance gene. Cells selected for resistance to the neomycin analog G418 formed aberrant colonies in monolayer cultures, marked by increased cell density and altered morphology. Transfected cells also grew readily in soft agar, producing colonies whose sizes correlated with E2A-HLF expression levels. Subclones expanded from colonies with high levels of the protein reproducibly formed tumors in nude mice and grew to higher plateau-phase cell densities in reduced-serum conditions than did parental NIH 3T3 cells. By contrast, NIH 3T3 cells expressing mutant E2A-HLF proteins that lacked either of the bipartite E2A trans-activation domains or the HLF leucine zipper domain failed to show oncogenic properties, including anchorage-independent cell growth. Thus, both of the E2A trans-activation motifs and the HLF leucine zipper dimerization domain are essential for the transforming potential of the chimeric E2A-HLF protein, suggesting a model in which aberrant regulation of the expression pattern of downstream target genes contributes to leukemogenesis.

3T3 Cells↗
PubMed1995

Stromal cell-mediated transcriptional regulation of the CD13/aminopeptidase N gene in leukemic cells.

CD13/aminopeptidase N (APN) is a cell surface metallopeptidase expressed by normal and leukemic myeloid cells, and by lymphoblasts in 5-10% of acute lymphoid leukemia (ALL) cases, previously classified as 'biphenotypic' or 'mixed-lineage' leukemias. In fresh cells from two early B-lineage, t(9;22)-positive, ALL cases that were CD13/APN-negative at diagnosis, high levels of CD13/APN expression were induced after 3 days of in vitro culture. Similarly, continuously growing cell lines established from these ALLs, KOPN-30bi and KOPN-57bi, expressed CD13/APN, but retained other phenotypic, cytochemical and molecular features of early B-lineage cells. After 7 days of culture on human bone marrow stromal layers or murine S17 stromal cells, levels of CD13/APN expression by the leukemic cell lines decreased by more than 4-fold. After 21 days of culture on stromal cells, CD13/APN became undetectable by flow cytometry; however, the original levels of expression were regained when the cell lines were cultured without stroma. A more moderate decrease in CD13/APN expression was also observed in the myeloid lines KG-1 and HL-60 during culture on stroma. Suppression of CD13/APN expression required contact with stroma, but did not depend on VLA-4-mediated adhesion. Surprisingly, the mechanism through which stromal cells down-regulated CD13/APN expression in leukemic cells involved suppression of transcription from the CD13/APN gene. Contact with stroma resulted in a 2-3-fold decrease in CD13/APN mRNA expression and near ablation of CD13/APN gene transcription in nuclear run-on assays. Thus, CD13/APN expression by leukemic cells is regulated by interactions with the bone marrow microenvironment. CD13/APN expression in some ALL at diagnosis could result from a block in the signal transduction pathways that cause its suppression by bone marrow stromal cells.

Bone Marrow Cells↗
PubMed1994

Chromosomal localization and cDNA cloning of the human DBP and TEF genes.

We have isolated cDNA and genomic clones and determined the human chromosome positions of two genes encoding transcription factors expressed in the liver and the pituitary gland: albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). Both proteins have been identified as members of the PAR (proline and acidic amino acid-rich) subfamily of bZIP transcription factors in the rat, but human homologues have not been characterized. Using a fluorescence in situ hybridization technique, the DBP locus was assigned to chromosome 19q13, and TEF to chromosome 22q13. Each assignment was confirmed by means of human chromosome segregation in somatic cell hybrids. Coding sequences of DBP and TEF, extending beyond the bZIP domain to the PAR region, were highly conserved in both human-human and interspecies comparisons. Conservation of the exon-intron boundaries of each bZIP domain encoding exon suggested derivation from a common ancestral gene. DBP and TEF mRNAs were expressed in all tissues and cell lines examined, including brain, lung, liver, spleen, and kidney. Knowledge of the human chromosome locations of these PAR proteins will facilitate studies to assess their involvement in carcinogenesis and other fundamental biological processes.

Amino Acid Sequence↗
PubMed1994

Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma.

The 2;5 chromosomal translocation occurs in most anaplastic large-cell non-Hodgkin's lymphomas arising from activated T lymphocytes. This rearrangement was shown to fuse the NPM nucleolar phosphoprotein gene on chromosome 5q35 to a previously unidentified protein tyrosine kinase gene, ALK, on chromosome 2p23. In the predicted hybrid protein, the amino terminus of nucleophosmin (NPM) is linked to the catalytic domain of anaplastic lymphoma kinase (ALK). Expressed in the small intestine, testis, and brain but not in normal lymphoid cells, ALK shows greatest sequence similarity to the insulin receptor subfamily of kinases. Unscheduled expression of the truncated ALK may contribute to malignant transformation in these lymphomas.

Amino Acid Sequence↗
PubMed1994

Cytogenetic abnormalities in two cases of neuroblastoma.

Neuroblastomas are common solid tumors in children. We report chromosome analysis of two neuroblastomas, each studied at diagnosis and at recurrence. The first case was a clinical stage D tumor which showed 45,X-Y, add(1)(p34),der(15)t(Y;15)(q11;p13), and double minutes on cytogenetic analysis at diagnosis. At recurrence, the same structural abnormalities were present along with a homogeneously staining region (hsr) at 8q22, 19p12, or 3p23 in each of three related clones. The hsr were shown to represent amplification of the N-myc gene by in situ hybridization. Cytogenetic analysis of the second tumor, stage D-S, showed 48-54,XX,der(1)add (1)(q41), +2, +7, +7, inv(9), +17, + mar. The lack of demonstrative involvement of 1p or visible evidence of gene amplification has also characterized the limited number of D-S specimens previously described, suggesting that stage D-S neuroblastoma indeed differs from stage D disease at the genetic level.

Abdominal Neoplasms↗
PubMed1994

Alterations in the PITSLRE protein kinase gene complex on chromosome 1p36 in childhood neuroblastoma.

p58cdc2L1, a protein kinase implicated in apoptotic signaling, is one of eight separate kinases encoded by three tandemly duplicated and linked genes, which we have termed PITSLRE A, B and C. One allele of this complex on chromosome 1 was either deleted or translocated in each of 18 neuroblastoma cell lines with cytogenetically apparent 1p alterations. A protein encoded by this locus, PITSLRE gamma 1, was absent in three of the lines and a smaller, apparently truncated, PITSLRE polypeptide was found in another line. These findings identify a novel gene complex on chromosome 1 that encodes a protein kinase subfamily. We suggest that the PITSLRE locus may harbour one or more tumour suppressor genes affected by chromosome 1p36 modifications in neuroblastoma.

Alleles↗
PubMed1994

Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

Animals↗
PubMed1994

DNA-binding specificity and trans-activating potential of the leukemia-associated E2A-hepatic leukemia factor fusion protein.

Hybrid transcription factors, resulting from gene fusions in the wake of chromosomal translocations, have been implicated in leukemogenesis, but their precise contributions to oncogenic conversion remain unclear. The E2A-HLF fusion gene, formed by a t(17;19)(q22;p13) in childhood pro-B-cell acute lymphoid leukemia, encodes a hybrid protein that contains the trans-activation domain of E2A (E12/E47) linked to the bZIP DNA-binding and dimerization domain of hepatic leukemia factor (HLF). Here we report that both HLF and E2A-HLF bind to a 10-bp consensus sequence, 5'-GTTACGTAAT-3', with a core dyad-symmetric motif characteristic of the bZIP scissors-grip model of DNA binding. A probe containing this sequence bound chimeric E2A-HLF proteins in nuclear extracts of a leukemic cell line (UOC-B1) containing the t(17;19), as demonstrated by complexes supershifted with antibodies specific for amino-terminal epitopes of E2A or carboxyl-terminal eptiopes of HLF. E2A-HLF functioned as a potent trans activator of reporter gene expression from a plasmid that contained the consensus DNA-binding sequence. Interestingly, wild-type HLF was restricted in its capacity to act as a trans activator, functioning in human fetal kidney cells but not HepG2 hepatocarcinoma cells or NIH 3T3 mouse fibroblasts. The ability of the E2A-HLF hybrid protein to bind DNA in a sequence-specific manner and trans activate the expression of artificial reporter genes suggests that it could subvert transcriptional programs that normally control the growth, differentiation, and survival of lymphoid progenitor cells.

3T3 Cells↗
PubMed1994

Splice-site mutation of the p53 gene in a family with hereditary breast-ovarian cancer.

Germline mutations within evolutionary conserved exons of the p53 gene predispose to tumor development in several familial cancer syndromes. We now report identification of a novel p53 mutation affecting the splice acceptor site of exon 6 in the germline DNA of a family with hereditary breast-ovarian cancer. This splice-site mutation, which results in omission of exon 6 and creates a frame-shift and premature stop codon in transcripts from the mutant allele, was found in seven family members--four of whom have developed breast, ovarian or choroid plexus tumors before age 35. Our finding suggests the need to examine the entire p53 gene for splice-site, frame-shift, and nonsense (as well as missense) mutations in families with early-onset hereditary breast and breast-ovarian cancers not linked to the BRCA1 gene on chromosome 17q. We propose that the term 'p53 familial cancer syndrome' be applied to clusters of tumors in families with documented germline p53 mutations, regardless of the histopathologic findings or pattern of tumor development.

Adolescent↗
PubMed1993

Coamplification of the CDK4 gene with MDM2 and GLI in human sarcomas.

The 34-kilodalton cyclin-dependent kinase, p34cdk4, is a major catalytic subunit of mammalian D-type cyclins, which act during the G1 phase of the cell cycle to enforce the decision of cells to enter S phase. A murine complementary DNA clone was used to clone the cognate human CDK4 gene, which was localized to human chromosome 12, band q13, by fluorescence in situ hybridization. Because this chromosomal band contains the GLI and MDM2 genes, which are frequently amplified in human sarcomas, we analyzed CDK4 copy number and expression in a panel of sarcoma cell lines. An osteosarcoma cell line, OsACL, manifested a 25-fold increased copy number of CDK4, amplified concordantly with both GLI and MDM2, whereas a rhabdomyosarcoma cell line, SJRH30, was found to have an amplicon that included CDK4 and GLI but not MDM2. CDK4 mRNA and protein were overexpressed in both cell lines, and nucleotide sequencing analysis indicated that the gene had not sustained mutations. These observations provide the first evidence for amplification of a gene encoding a cell division cycle protein kinase, complement recent data indicating that genes encoding D-type cyclins are targets of chromosomal rearrangement and gene amplification in tumor cells, and suggest that CDK4 amplification might contribute to oncogenesis.

Base Sequence↗
PubMed1993

Clinical significance of CD34 expression in childhood acute lymphoblastic leukemia.

The CD34 antigen was detected on > or = 10% of the blast cells in 235 (70%) of 335 cases of newly diagnosed childhood acute lymphoblastic leukemia (ALL) treated in two consecutive chemotherapy trials. By immunophenotype, the distribution of positive cases favored early pre-B ALL (83%; n = 180) followed by pre-B ALL (61%; n = 89) and then T-cell ALL (46%; n = 61) (P < .001). Among the B-lineage cases, CD34 expression was significantly associated with favorable presenting features: age 1 to 10 years, white race, absence of central nervous system (CNS) leukemia, low serum lactate dehydrogenase level, CD10 expression, and leukemic cell hyperdiploidy (> 50 chromosomes or DNA index > or = 1.16). Event-free survival was clearly superior for patients with CD34+ leukemia (P = .01), with an estimated 83% +/- 6% (SE) of the cohort remaining free of adverse events at 5 years post diagnosis, as compared to 63% +/- 10% of the group without this feature. Multivariate analysis showed that the prognostic influence of the antigen was independent of age, leukocyte count, and other well-recognized factors, suggesting that it would add discriminatory power to current systems of risk assignment. Findings in T-cell ALL were the reverse: CD34 expression showed positive correlations with initial CNS leukemia and CD10 negativity but not with any good-risk presenting characteristics. Log-rank analysis indicated no adverse effect on treatment outcome by CD34 antigen expression, although additional patients with need to be studied to obtain a definitive answer. The opposed clinical associations of CD34 expression in B- and T-lineage ALL may reflect fundamental biologic differences between these leukemia species.

Antigens, CD↗
PubMed1993

Inactivation of the NF1 gene in human melanoma and neuroblastoma cell lines without impaired regulation of GTP.Ras.

The NF1 gene, which is altered in patients with type 1 neurofibromatosis, encodes neurofibromin, a protein whose GTPase-activating function can negatively regulate GTP-Ras by accelerating its conversion to inactive GDP-Ras. In schwannoma cell lines from patients with neurofibromatosis, loss of neurofibromin was previously shown to be associated with impaired regulation of GTP-Ras. Our analysis of other neural crest-derived tumor cell lines has shown that some melanoma and neuroblastoma cell lines established from tumors occurring in patients without neurofibromatosis contain reduced or undetectable levels of neurofibromin, with concomitant genetic abnormalities of the NF1 locus. In contrast to the schwannoma cell lines, GTP-Ras was appropriately regulated in the melanoma and neuroblastoma lines that were deficient in neurofibromin, even when c-H-ras was overexpressed in the lines. These results demonstrate that some neural crest tumors not associated with neurofibromatosis have acquired somatically inactivated NF1 genes and suggest a tumor-suppressor function for neurofibromin that is independent of Ras GTPase activation.

Blotting, Northern↗
PubMed1993

Correlation between morphologic and other prognostic markers of neuroblastoma. A study of histologic grade, DNA index, N-myc gene copy number, and lactic dehydrogenase in patients in the Pediatric Oncology Group.

BACKGROUND: Histologic grades (HG), N-myc (NM) gene copy number, DNA index (DI), and serum lactic dehydrogenase (LDH) have been shown to be related to prognosis in neuroblastoma. The relationship between HG and nonmorphologic prognostic markers has not been investigated previously. METHODS: Each prognostic marker was determined independently and without the knowledge of clinical features and outcome by different investigators in 275 (HG), 96 of 275 (DI), 94 of 275 (NM), and 224 of 275 patients (LDH) with neuroblastoma by methods described previously. Patients younger than 2 years of age were included in the analysis for DI. Patients of all ages were included in the analysis of HG, NM, and LDH. RESULTS: A statistically significant association of low HG (1 and 2) was found with DI of more than 1 (hyperdiploid), single copy of NM gene per haploid genome, and an LDH of less than 1500 IU/l (P value for each, < 0.001), factors that are associated with better prognosis. High HG was associated with DI of 1 (diploidy), amplified NM gene, and an LDH of 1500 or more, factors that are associated with aggressive behavior. CONCLUSION: The value of HG is strengthened by its statistically significant association with features that reflect tumor cell biology of neuroblastoma. In view of the tissue sample size required for determination of HG, consideration should be given to obtaining such a sample in as many patients as is feasible if there is no contraindication to surgery. Nonmorphologic prognostic markers, when used in concert with HG, would provide a basis for individualized risk-specific therapy of this disease.

Adolescent↗
PubMed1993

Cell transformation by ras and regulation of its protein product.

We are studying the biological activity and regulation of mammalian Ras protein in tumours and in physiological signalling. We have shown that GAP (the GTPase-activating protein) is a potent negative regulator of normal Ras in cells. Reduction or loss of the NF1 gene product neurofibromin, in association with genetic abnormalities of the NF1 locus, has been identified in schwannoma cell lines from patients with neurofibromatosis and in melanoma and neuroblastoma lines from patients without neurofibromatosis. Although loss of neurofibromin in the schwannoma lines was associated with a high proportion of normal Ras protein in the active GTP-bound state, Ras-GTP appeared to be appropriately regulated in the melanoma and neuroblastoma lines, which contain normal levels of GAP. Therefore the GTPase-activating activity of neurofibromin is not essential for negative regulation of Ras in some cell types and the putative tumour suppressor function of neurofibromin in such cell types is independent of its GTPase-activating activity. Mitogen activation of Ras in fibroblasts is mediated primarily by exchange factors, which probably interact with a region on the Ras protein distinct from the region required for interaction with GAP. Multiple full-length cDNAs have identified a mouse gene whose products are related to yeast CDC25 guanine nucleotide exchange factor.

Animals↗